Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M
Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M
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DOI：
10.1074/jbc.m110.204057
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发表时间：
2011-05-06
影响因子：
4.8
通讯作者：
Gaur, Rajesh K.
中科院分区：
文献类型：
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作者：
Dery, Kenneth J.;Gaur, Shikha;Gaur, Rajesh K.
癌胚抗原相关细胞粘附分子-1（CEACAM 1）在多种细胞类型中表达，并与致癌作用有关。CEACAM 1前体mRNA的选择性剪接产生两种胞质结构域剪接变体，其特征在于外显子7的包含（L-同种型）或排除（S-同种型）。在这里，我们表明CEACAM 1前mRNA的选择性剪接是由位于外显子7的新顺式元件调节的。我们报告存在三个外显子调控元件，导致列入或排除外显子7 CEACAM 1 mRNA在ZR 75乳腺癌细胞。异源剪接报告分析表明，真实的选择性剪接机制的维护是独立的CEACAM 1内含子序列上下文。我们发现，这些外显子调控元件的强制表达可以改变HEK-293细胞中CEACAM 1的剪接。使用RNA亲和层析，三个成员的异质核核糖核蛋白家族（hnRNP L，hnRNP A1，和hnRNP M）进行了鉴定。hnRNPL和hnRNPA 1的RNA免疫沉淀分别显示位于外显子7的中央和3'的结合基序。在HEK-293细胞中通过RNAi耗尽hnRNP A1或L促进外显子7包含，而过表达导致可变外显子的排除。相反，hnRNP M的过表达显示外显子7包含和CEACAM 1-L mRNA的产生。最后，MDA-MB-468细胞中应激诱导的hnRNP A1的细胞质积累动态地改变CEACAM 1-S：CEACAM 1：L比率，有利于L-亚型。因此，我们已经阐明了在CEACAM 1的选择性剪接调控中控制剪接位点识别机制的分子因素。
Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S: CEACAM1: L ratio in favor of the L-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.